臺灣博碩士論文加值系統

鮑氏不動桿菌(Acinetobacter baumannii)，屬於革蘭氏陰性球桿菌，於臨床上易造成許多院內感染症，包括肺炎、菌血症、腦膜炎及泌尿道感染等。近年於國內各大醫院極易從臨床病人身上分離出多重抗藥性鮑氏不動桿菌(multiple-drug resistant Acinetobacter baumannii，簡稱MDRAB)，此細菌對大部分抗生素均呈現抗藥性。針對MDRAB的感染，粘菌素(colistin) 被發現具有治療MDRAB感染的能力；然而，隨著藥物的廣泛使用，臨床上的MDRAB也逐漸產生對抗粘菌素的能力。為了找尋鮑氏不動桿菌與粘菌素抗藥性相關的基因，本研究首先建構多重抗藥性鮑氏不動桿菌表現基因庫，並嘗試篩選出可能與粘菌素抗藥性相關的基因。之後我們經由上述表現基因庫篩選到三個與粘菌素抗藥性相關基因，它們分別為A1S_0008、A1S_3219及A1S_3220。經實驗證實這三個基因(A1S_0008、A1S_3219及A1S_3220)之mRNA表現量在粘菌素誘導抗藥菌株較野生株高。此外，經由相關文獻得知細菌能透過lipid A phosphoethanolamine transferase改變陽離子粘菌素與脂多醣體鍵結能力，造成對粘菌素產生抗性。因此我們針對鮑氏不動桿菌之lipid A phosphoethanolamine transferase (A1S_2752)及其下游基因(A1S_2751及A1S_2750)進行分析。透過細菌體內mRNA表現量得知上述三個基因在粘菌素誘導抗藥菌株之mRNA表現量較高，另外在大部分粘菌素誘導抗藥臨床菌株也觀察到同樣的現象。接下來本研究將進一步證實上述六個基因與粘菌素抗藥性的相關性，期許能夠找出新的粘菌素抗藥機轉並對未來藥物研發有所貢獻。

Acinetobacter baumannii is a gram-negative bacterium which causes serious nosocomial infections, such as pneumonia, bacteremia, meningitis, and urinary tract infections. There are many new strains of A. baumannii recently isolated and proven to be resistant to clinically-used antibiotics; they are called multiple-drug resistant A. baumannii (MDRAB). At present, colistin can effectively treat A. baumannii; however, there are sporadically colistin-resistant strains found clinically. Therefore, the aim of our study is to investigate the genes associated with colistin resistance. In order to investigate genes related to colistin resistance, we constructed an expression library from a colistin resistant clinical strain. After screened the expression library, we isolated three genes (A1S_0008, A1S_3219 and A1S_3220) relative to colistin resistance. The quantitative PCR results indicated that mRNA expression levels of these genes in artificially induced colistin-resistant strains were significantly higher than those of the wild-type strains. Furthermore, according to some review papers, the development of colistin resistance may be due to the change in the binding affinity between cationic colistin and lipopolysaccharide. Thus, we want to examine whether lipid phosphoethanolamine transferase gene (A1S_2752) and its downstream genes (A1S_2751 and A1S_2750) are associated with colistin resistance. The expression levels of these genes were analyzed and we found that their mRNA expression levels in artificially induced colistin-resistant strains were also higher than those of the wild-type strains. The relationship between these six genes and colistin resistance can be further analyzed. We hope this research would be the basis of eliminating drug resistant strains, and promote the further development of new antimicrobial agents in the future.

中文摘要 I
Abstract II
第一章 前言 1
1.1 不動桿菌屬(Acinetobacter)之命名及特性 1
1.2 鮑氏不動桿菌(A. baumannii)的致病性及傳染途徑 1
1.3 鮑氏不動桿菌感染症的治療 2
1.4 多重抗藥性鮑氏不動桿菌的興起 2
1.5 粘菌素(colistin)的特性與臨床應用 3
1.6 粘菌素(colistin)抗藥基因之研究 4
1.7 幫浦系統與細菌抗藥基因之相關研究 4
1.8 研究目的 5
第二章 材料 6
2.1培養基 6
2.2本實驗所使用之大腸桿菌菌株 7
2.3本實驗所使用之鮑氏不動桿菌菌株 8
2.4核酸引子 9
2.5抗生素 10
2.6質體 10
2.7限制酶 12
第三章 方法 13
第一部分 從MDRAB表現基因庫篩選出與粘菌素抗藥性相關之基因 13
3.1.1抗生素感受性試驗 13
3.1.2 MDRAB表現基因庫之建構 13
3.1.3生體切除(In vivo excision) 14
3.1.4粘菌素抗藥基因的大腸桿菌XLOLR菌株篩選 15
3.1.5萃取質體DNA 15
3.1.6製備熱休克勝任細胞 16
3.1.7熱休克進行再轉型作用 16
3.1.8聚合酶連鎖反應(PCR)及限制酶切割反應進行篩選確認 17
3.1.9 DNA定序及資料庫比對分析 17
第二部分 以生物資訊工具找出與粘菌素抗藥性相關之基因 18
第三部分 粘菌素(colistin)抗藥基因選殖部分 18
3.3.1繼代培養及誘導抗藥菌株試驗 18
3.3.2細菌生長曲線測試 19
3.3.3萃取細菌染色體DNA 19
3.3.4以AP-PCR方式確認細菌型別 20
3.3.5 TA選殖 20
3.3.6純化total RNA 21
3.3.7以聚合酶鏈鎖反應確認無殘留之DNA 22
3.3.8反轉錄聚合酶連鎖反應(RT-PCR) 22
3.3.9試管內破壞標的基因 22
3.3.10以電穿孔(electroporation)進行生體破壞 23
電穿孔之勝任細胞的製備 24
生體同源互換(Homologus recombination)及電穿孔之條件 24
第四章 結果 25
第一部分 從MDRAB表現基因庫篩選出與粘菌素抗藥性相關之基因 25
4.1.1細菌抗生素感受性試驗結果 25
4.1.2 MDRAB36788及AB2383CR表現基因庫建構結果 25
4.1.3經由生體切除(In vivo excision)篩選粘菌素(colistin)抗藥基因之結果 26
第二部分 以生物資訊工具找出與粘菌素抗藥性相關基因之分析 27
第三部分 粘菌素(colistin)抗藥基因選殖及功能分析結果 28
4.3.1鮑氏不動桿菌野生株誘導粘菌素抗藥菌株(CR)試驗結果 28
4.3.2 ATCC17978與17978CR生長曲線(Growth curve)結果 28
4.3.3 AP-PCR分型結果 28
4.3.4 TA選殖與點突變分析結果 28
4.3.5 RNA表現量分析結果 29
4.3.6試管內破壞(in vitro knockout)標的基因之結果 30
4.3.7構築生體內同源破壞(In vivo homologous recombination)突變株之結果 30
4.3.8構築部分基因缺失生體內同源破壞突變株之結果 31
第五章 討論與未來展望 32
第六章 圖表 36
表一、鮑氏不動桿菌菌株及實驗菌株對於粘菌素之感受性分析結果 36
表二、MDRAB表現基因庫篩選與粘菌素抗藥基因相關之基因列表 37
表三、粘菌素抗藥標的基因之列表 38
圖一、pBK-CMV表現載體及生體切除(in vivo excision)質體示意圖 39
圖二：利用PCR確認篩選表現基因庫體內是否含有嵌入之多重抗藥性鮑氏不動桿菌之3~5 kbp DNA片段 40
圖三、ATCC17978及17978CR菌株生長曲線圖 41
圖四、AP-PCR確認細菌之型別 42
圖五、ATCC17978及17978CR之A1S_2750、A1S_2751及A1S_2752 RNA表現量之分析 43
圖六、ATCC17978及17978CR之A1S_3219、A1S_3220及A1S_0008 RNA 44
圖七、臨床菌株及其誘導抗藥菌株之RNA表現量分析 45
圖八、試管內破壞 (In vitro knockout ) 標的基因之結果分析 46
圖九、生體內同源破壞 (In vivo knockout ) 標的基因之結果分析 47
圖十、部分基因缺失同源破壞之模式 48
圖十一、部分基因缺失同源破壞之結果分析 49
第七章 參考文獻 50

1.Adams, M. D., G. C. Nickel, S. Bajaksouzian, H. Lavender, A. R. Murthy, M. R. Jacobs, and R. A. Bonomo. 2009. Resistance to colistin in Acinetobacter baumannii associated with mutations in the PmrAB two-component system. Antimicrob Agents Chemother 53:3628-34.
2.Baraibar, J., H. Correa, D. Mariscal, M. Gallego, J. Valles, and J. Rello. 1997. Risk factors for infection by Acinetobacter baumannii in intubated patients with nosocomial pneumonia. Chest 112:1050-4.
3.Barbe, V., D. Vallenet, N. Fonknechten, A. Kreimeyer, S. Oztas, L. Labarre, S. Cruveiller, C. Robert, S. Duprat, P. Wincker, L. N. Ornston, J. Weissenbach, P. Marliere, G. N. Cohen, and C. Medigue. 2004. Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res 32:5766-79.
4.Baumann, P. 1968. Isolation of Acinetobacter from soil and water. J Bacteriol 96:39-42.
5.Bengoechea, J. A., and M. Skurnik. 2000. Temperature-regulated efflux pump/potassium antiporter system mediates resistance to cationic antimicrobial peptides in Yersinia. Mol Microbiol 37:67-80.
6.Bergogne-Berezin, E., and K. J. Towner. 1996. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev 9:148-65.
7.Bosso, J. A., C. A. Liptak, D. K. Seilheimer, and G. M. Harrison. 1991. Toxicity of colistin in cystic fibrosis patients. DICP 25:1168-70.
8.Bou, G., G. Cervero, M. A. Dominguez, C. Quereda, and J. Martinez-Beltran. 2000. PCR-based DNA fingerprinting (REP-PCR, AP-PCR) and pulsed-field gel electrophoresis characterization of a nosocomial outbreak caused by imipenem- and meropenem-resistant Acinetobacter baumannii. Clin Microbiol Infect 6:635-43.
9.Bratu, S., D. Landman, D. A. Martin, C. Georgescu, and J. Quale. 2008. Correlation of antimicrobial resistance with beta-lactamases, the OmpA-like porin, and efflux pumps in clinical isolates of Acinetobacter baumannii endemic to New York City. Antimicrob Agents Chemother 52:2999-3005.
10.Chang, P. Y., P. R. Hsueh, P. S. Wu, P. C. Chan, T. T. Yang, C. Y. Lu, L. Y. Chang, J. M. Chen, P. I. Lee, C. Y. Lee, and L. M. Huang. 2007. Multidrug-resistant Acinetobacter baumannii isolates in pediatric patients of a university hospital in Taiwan. J Microbiol Immunol Infect 40:406-10.
11.Chen, C. M., P. Y. Liu, S. C. Ke, H. J. Wu, and L. T. Wu. 2009. Investigation of carbapenem-resistant Acinetobacter baumannii isolates in a district hospital in Taiwan. Diagn Microbiol Infect Dis 63:394-7.
12.Conway, S. P., M. N. Pond, A. Watson, C. Etherington, H. L. Robey, and M. H. Goldman. 1997. Intravenous colistin sulphomethate in acute respiratory exacerbations in adult patients with cystic fibrosis. Thorax 52:987-93.
13.Damier-Piolle, L., S. Magnet, S. Bremont, T. Lambert, and P. Courvalin. 2008. AdeIJK, a resistance-nodulation-cell division pump effluxing multiple antibiotics in Acinetobacter baumannii. Antimicrob Agents Chemother 52:557-62.
14.Dean, C. R., M. A. Visalli, S. J. Projan, P. E. Sum, and P. A. Bradford. 2003. Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1. Antimicrob Agents Chemother 47:972-8.
15.Devaud, M., F. H. Kayser, and B. Bachi. 1982. Transposon-mediated multiple antibiotic resistance in Acinetobacter strains. Antimicrob Agents Chemother 22:323-9.
16.Dijkshoorn, L., A. Nemec, and H. Seifert. 2007. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol 5:939-51.
17.Fernandez-Reyes, M., M. Rodriguez-Falcon, C. Chiva, J. Pachon, D. Andreu, and L. Rivas. 2009. The cost of resistance to colistin in Acinetobacter baumannii: a proteomic perspective. Proteomics 9:1632-45.
18.Gerner-Smidt, P., I. Tjernberg, and J. Ursing. 1991. Reliability of phenotypic tests for identification of Acinetobacter species. J Clin Microbiol 29:277-82.
19.Gunn, J. S., and S. I. Miller. 1996. PhoP-PhoQ activates transcription of pmrAB, encoding a two-component regulatory system involved in Salmonella typhimurium antimicrobial peptide resistance. J Bacteriol 178:6857-64.
20.Gunn, J. S., S. S. Ryan, J. C. Van Velkinburgh, R. K. Ernst, and S. I. Miller. 2000. Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium. Infect Immun 68:6139-46.
21.Haagensen, J. A., M. Klausen, R. K. Ernst, S. I. Miller, A. Folkesson, T. Tolker-Nielsen, and S. Molin. 2007. Differentiation and distribution of colistin- and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms. J Bacteriol 189:28-37.
22.Hirata, T., A. Saito, K. Nishino, N. Tamura, and A. Yamaguchi. 2004. Effects of efflux transporter genes on susceptibility of Escherichia coli to tigecycline (GAR-936). Antimicrob Agents Chemother 48:2179-84.
23.Hsueh, P. R., L. J. Teng, C. Y. Chen, W. H. Chen, C. J. Yu, S. W. Ho, and K. T. Luh. 2002. Pandrug-resistant Acinetobacter baumannii causing nosocomial infections in a university hospital, Taiwan. Emerg Infect Dis 8:827-32.
24.Joly-Guillo, M. L., E. Vallee, E. Bergogne-Berezin, and A. Philippon. 1988. Distribution of beta-lactamases and phenotype analysis in clinical strains of Acinetobacter calcoaceticus. J Antimicrob Chemother 22:597-604.
25.Kampfer, P., I. Tjernberg, and J. Ursing. 1993. Numerical classification and identification of Acinetobacter genomic species. J Appl Bacteriol 75:259-68.
26.Kapil, A., S. Gulati, V. Goel, L. Kumar, R. Krishnan, and V. Kochupillai. 1998. Outbreak of nosocomial Acinetobacter baumannii bacteremia in a high risk ward. Med Oncol 15:270-4.
27.Keeney, D., A. Ruzin, F. McAleese, E. Murphy, and P. A. Bradford. 2008. MarA-mediated overexpression of the AcrAB efflux pump results in decreased susceptibility to tigecycline in Escherichia coli. J Antimicrob Chemother 61:46-53.
28.Kuo, L. C., C. J. Yu, L. N. Lee, J. L. Wang, H. C. Wang, P. R. Hsueh, and P. C. Yang. 2003. Clinical features of pandrug-resistant Acinetobacter baumannii bacteremia at a university hospital in Taiwan. J Formos Med Assoc 102:601-6.
29.Leahy, J. G., J. M. Jones-Meehan, E. L. Pullias, and R. R. Colwell. 1993. Transposon mutagenesis in Acinetobacter calcoaceticus RAG-1. J Bacteriol 175:1838-40.
30.Li, J., R. L. Nation, R. W. Milne, J. D. Turnidge, and K. Coulthard. 2005. Evaluation of colistin as an agent against multi-resistant Gram-negative bacteria. Int J Antimicrob Agents 25:11-25.
31.Li, J., C. R. Rayner, R. L. Nation, R. J. Owen, D. Spelman, K. E. Tan, and L. Liolios. 2006. Heteroresistance to colistin in multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 50:2946-50.
32.Macfarlane, E. L., A. Kwasnicka, M. M. Ochs, and R. E. Hancock. 1999. PhoP-PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance. Mol Microbiol 34:305-16.
33.Mai, X., and M. W. Adams. 1994. Indolepyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus. A new enzyme involved in peptide fermentation. J Biol Chem 269:16726-32.
34.Marceau, M., F. Sebbane, F. Ewann, F. Collyn, B. Lindner, M. A. Campos, J. A. Bengoechea, and M. Simonet. 2004. The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence. Microbiology 150:3947-57.
35.Marchand, I., L. Damier-Piolle, P. Courvalin, and T. Lambert. 2004. Expression of the RND-type efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS two-component system. Antimicrob Agents Chemother 48:3298-304.
36.McPhee, J. B., M. Bains, G. Winsor, S. Lewenza, A. Kwasnicka, M. D. Brazas, F. S. Brinkman, and R. E. Hancock. 2006. Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg2+-induced gene regulation in Pseudomonas aeruginosa. J Bacteriol 188:3995-4006.
37.Moskowitz, S. M., R. K. Ernst, and S. I. Miller. 2004. PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A. J Bacteriol 186:575-9.
38.Pagel, J. E., and P. L. Seyfried. 1976. Numerical taxonomy of aquatic Acinetobacter isolates. J Gen Microbiol 96:220-32.
39.Peleg, A. Y., J. Adams, and D. L. Paterson. 2007. Tigecycline Efflux as a Mechanism for Nonsusceptibility in Acinetobacter baumannii. Antimicrob Agents Chemother 51:2065-9.
40.Ribera, A., I. Roca, J. Ruiz, I. Gibert, and J. Vila. 2003. Partial characterization of a transposon containing the tet(A) determinant in a clinical isolate of Acinetobacter baumannii. J Antimicrob Chemother 52:477-80.
41.Ross, S., J. R. Puig, and E. A. Zaremba. 1959. Colistin: some preliminary laboratory and clinical observations in specific gastroenteritis in infants and children. Antibiot Annu 7:89-100.
42.Ruzin, A., D. Keeney, and P. A. Bradford. 2007. AdeABC multidrug efflux pump is associated with decreased susceptibility to tigecycline in Acinetobacter calcoaceticus-Acinetobacter baumannii complex. J Antimicrob Chemother 59:1001-4.
43.Ruzin, A., M. A. Visalli, D. Keeney, and P. A. Bradford. 2005. Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae. Antimicrob Agents Chemother 49:1017-22.
44.Scerpella, E. G., A. R. Wanger, L. Armitige, P. Anderlini, and C. D. Ericsson. 1995. Nosocomial outbreak caused by a multiresistant clone of Acinetobacter baumannii: results of the case-control and molecular epidemiologic investigations. Infect Control Hosp Epidemiol 16:92-7.
45.Schwartz, B. S., M. R. Warren, F. A. Barkley, and L. Landis. 1959. Microbiological and pharmacological studies of colistin sulfate and sodium colistinmethanesulfonate. Antibiot Annu 7:41-60.
46.Seifert, H., A. Strate, and G. Pulverer. 1995. Nosocomial bacteremia due to Acinetobacter baumannii. Clinical features, epidemiology, and predictors of mortality. Medicine (Baltimore) 74:340-9.
47.Siddiqui, M. A., S. Fujiwara, and T. Imanaka. 1997. Indolepyruvate ferredoxin oxidoreductase from Pyrococcus sp. KOD1 possesses a mosaic structure showing features of various oxidoreductases. Mol Gen Genet 254:433-9.
48.Singer, J. T., and W. R. Finnerty. 1984. Insertional specificity of transposon Tn5 in Acinetobacter sp. J Bacteriol 157:607-11.
49.Tamayo, R., S. S. Ryan, A. J. McCoy, and J. S. Gunn. 2002. Identification and genetic characterization of PmrA-regulated genes and genes involved in polymyxin B resistance in Salmonella enterica serovar typhimurium. Infect Immun 70:6770-8.
50.Towner, K. J. 1983. Transposon-directed mutagenesis and chromosome mobilization in Acinetobacter calcoaceticus EBF65/65. Genet Res 41:97-102.
51.Tran, A. X., J. D. Whittimore, P. B. Wyrick, S. C. McGrath, R. J. Cotter, and M. S. Trent. 2006. The lipid A 1-phosphatase of Helicobacter pylori is required for resistance to the antimicrobial peptide polymyxin. J Bacteriol 188:4531-41.
52.Vashist, J., and M. R. Rajeswari. 2006. Structural investigations on novel porin, OmpAb from Acinetobacter baumannii. J Biomol Struct Dyn 24:243-53.
53.Wang, W. B., I. C. Chen, S. S. Jiang, H. R. Chen, C. Y. Hsu, P. R. Hsueh, W. B. Hsu, and S. J. Liaw. 2008. Role of RppA in the regulation of polymyxin b susceptibility, swarming, and virulence factor expression in Proteus mirabilis. Infect Immun 76:2051-62.
54.Wright, W. W., and H. Welch. 1959. Chemical, biological and clinical observations on colistin. Antibiot Annu 7:61-74.